The mouse shares 99% of the genes with humans and is an ideal research model for enhancing our understanding of human biology and disease and for testing potential therapeutics. Our ability to utilize the mouse model is limited by the number of mouse mutants and disease models (3000 - 4000) that are available to the research and pharmaceutical community. We have developed a new transposon system, called pB, which can be used to efficiently mutate a large number of genes in mice and other mammalian systems. This system has the following advantageous characters: 1. pB transposes efficiently in the mouse germline and can rapidly produce a large number of single pB insertion mutant strains;2 Disrupted genes can be easily identified by PCR and sequencing;3. pB insertions favor genes and have a wide genomic distribution;4. pB insertions disrupt gene function when inserted into genes, and produce phenotypes similar to mutants generated by traditional knockout methods;5. pB can carry large DNA fragments, e.g., an Act-RFP marker and a LacZ reporter, which can report gene expression patterns;6. The resulting heterozygous or homozygous mutant animals and their wild-type siblings can be visually distinguished from each other without PCR or Southern using reporters such as Act-RFP;7. Mutant animals are directly produced by simple breeding, which bypasses costly and challenging traditional techniques involving ES cells and surgery;8. Visible genetic markers are employed in the breeding scheme to further improve efficiency and reduce cost;9. All mutants are generated in an identical genetic background;10. pB insertions can be precisely excised to revert the mutant phenotypes. We propose to utilize this pB insertional mutagenesis system and the cost-effective animal facility in Shanghai, China to produce 100,000 independent single pB insertion mutant strains in C57BL/6 mice in five years. We will determine the insertion sites, establish a database, and generate a comprehensive mouse KO resource which consists of frozen mutant embryos or sperm for approximately 20,000 unique genes or genetic loci. We estimate that the costs for the production and characterization of an independent insertion strain and for the storage of that strain are approximately $148 and $760, respectively. This comprehensive mouse mutant resource will significantly aid our ability to understand biology and improve human health.